1. Field of the Invention
This invention relates to novel physiologically active peptides possessing cell adhesion inhibitory activity. These novel physiologically active peptides are useful as therapeutic agents for various diseases which are believed to involve cell adhesion factors and/or ligand molecules to the cell adhesion factors, such as cancer, inflammation including rheumatism, and fibrosis.
2. Related Background Art
Numerous remedies have been tried in the treatment of cancer, and notable therapeutic effects have come to be seen in a number of cancers. Under the present circumstances, methods of treatment for the prevention of cancer metastasis, however, have not yet been established.
When the patient has been diagnosed as having a cancer, principal treatments include a surgical treatment, radiotherapy and chemotherapy. In a case where the patient complains of obvious abnormality, it is almost certain that the patient has the condition of a progressive cancer; therefore, the treatment is not easy. It is no exaggeration that what brings such condition may be, in short, through metastatic growth of the cancer. The prognosis of the patient is almost governed by the degree of cancer metastatis (Welch, et al., Intern. J. Cancer, 43, 449, 1989). Unless the metastatic growth of a cancer can be suppressed, it will be extremely difficult to control the cancer once it has developed. In a situation where a stomach cancer has progressed, it is likely that the cancer has already migrated to the lymph nodes, liver, lung and other organs. The same is true with a breast cancer, and the fact of the matter is that the progressive cancer has migrated not only to its peripheral lymph nodes, but also to the bone marrow or the lung. Particularly, it is the degree of lymphogenous metastasis that determines the prognosis of epithelial cancers (Shigeru Tsukagoshi, Chemistry for the Conquest of Cancer. Pharmacia Review, No. 6: The Pharmaceutical Society of Japan).
Metastasis of a cancer consists of complex steps: beginning with detachment of cancer cells from the primary tumor mass and their invasion into peripheral tissues, and leading to their proliferation in the migrated tissues. Elements, both in the cancer cells and the host, influence these steps. In the former cells, the primary tumor mass comprises a group of cells of ununiformity which exhibit cellular characters differing with respect to proliferation capability, drug susceptibility, immunogenicity, and morphology as a result of incessant gene alternation: the steps involve the elements such as the propensities of cells to migrate and to invade the peripheral tissues, and production of autonomous proliferation factors. The elements on the host side are thought to be physical and anatomic conditions in blood, defense mechanisms by immunocompetent cells, adhesive interactions between vascular endothelial cells and platelets, effects of growth factors from the peripheral tissues of metastases, and the like.
It is believed that during a series of steps forming cancer metastases, the expression and regulation of metastatic characters of cancer cells is determined under the influence of various biological components containing normal cells, cancer cells themselves and extracellular matrices in the host.
Adhesion between cancer cells or between cancer cells and normal cells is mediated by cell adhesion molecules that are expressed on the surfaces of cells. The cadherin family (Masatoshi Takechi, Molecular Basis for Tissue Construction-the Role of Cadherin in Cell Recognition, Biochemistry, 59, 1, 1987), the immunoglobulin family and the selectin family (Springer, T. A., Nature, 346, 425, 1990) are known as cell adhesion molecules. Adherent molecules on the surfaces of cells, including the integrin family, are known to be involved in adhesion between cancer cells and extracellular matrices including glycoproteins such as fibronectin, laminin and collagen, and proteoglycans to which heparan sulfate or chondroitin sulfate is bound. These extracellular matrix molecules are deeply involved in the metastasis of cancer.
It has been shown that when cells are transformed to malignancy by oncogenic viruses or carcinogens, the synthesis of extracellular matrix molecules, such as fibronectins, and their extracellular accumulation diminishes (Yamada, K. M. and Olden, K., Nature 275, 179, 1978), and that the level of expression of integrin molecules diminishes, or adhesiveness to fibronectins is reduced (Plantfaber, L. C. and Hynes, R. O., Cell 56, 281, 1989). It has also been reported that both fibronectin and laminin promote the adhesion of cancer cells as well as their motility (McCarthy, J. B., J. Cell Biol., 98, 1474, 1984).
Fibronectin and laminin molecules have domain structures through which many functions are dispersed within their molecules. While the structures have been elucidated by techniques, biochemically or through genetic engineering, the presence of cell adhesion sites and receptors thereto have been discovered. The RGD sequence within the cell-binding domain of fibronectin has been identified to be a cell recognition sequence (Pierschbacher, M. D., Nature 309, 30, 1984); it has been determined that the sequence also exists and functions in many cell adhesion related molecules and that its cell receptor is xcex15xcex21 integrin receptor (Pytela, R., Cell 40, 191, 1985).
For the reasons stated above, as adhesive interactions between the cell adhesion molecules and cells have become apparent, attempts are being made to make use of the peptides that antagonize cell adhesion as anti-metastatics of cancer.
Humphries et al. discloses that GRGDS peptide, which functions as a cell adhesion signal of fibronectin, inhibits the experimental metastasis of melanoma cells to the lung (Humphries, M. J., Science 233, 467, 1986).
However, there are many problems to be solved before the peptide can be put to practical use as drug: for example, the rate of clearance of the peptide from the circulation following administration is fast, and it is liable to be decomposed by enzyme (Saiki, I., et al., Jpn. J. Cancer Res., 84, 558, 1993).
Fukai et al. reports that when a 30-kDa fragment derived from the Hep2 domain of fibronectin (referred to as xe2x80x9cHep2xe2x80x9d hereafter) is denatured with urea, it acquires a strong cell adhesion inhibitory activity (Fukai, F., et al., Biochemica. Biophys. Res. Commun., 220, 394, 1996).
The reference, however, does not disclose which amino acid sequence results in the cell adhesion inhibitory activity by Hep2. Further, because Hep2 consists of 271 amino acid residues, it is undesirable that Hep2 itself be put to practical use as drug for the following reasons among others: the synthesis and preparation of Hep2 is difficult owing to its large molecular weight, and there is the possibility that it exhibits an unfavorable physiological activity since it contains amino acid sequences unrelated to the cell adhesion inhibitory activity.
As stated above, searches for inhibitory substances for cancer metastasis are indispensable for the control of cancers; however, under the present circumstances no substance has yet been obtained that would serve as a specific remedy and thus has been put to practical use.
In light of the aforementioned circumstances, this invention has been made, and it aims at providing novel physiologically active peptides possessing cell adhesion inhibitory activity.
Furthermore, it is an object of the invention to provide anti-metastatics comprising the novel physiologically active peptides.
The present inventors have diligently pursued further research with the aim toward solving the aforementioned problems and have successfully discovered novel peptides possessing an antagonistic action against the action of cell adhesion factors that promote diseases such as cancer, inflammation and fibrosis and/or against the action of ligand molecules to the cell adhesion factors: the peptides containing a consensus amino acid sequence of Thr Ile Thr and comprising amino acid sequences set forth in SEQ ID NOs: 2, 3, and 7-9 in the Sequence Listing.
These peptides are shorter in segment than Hep2 and possess a strong cell adhesion inhibitory activity without being subjected to denaturation treatment such as urea treatment.
The physiologically active peptides of this invention are also analogous to specific sequences of the amino acid sequence of human fibronectin protein.
Specifically, the invention preferably relates to a physiologically active peptide possessing cell adhesion inhibitory activity and comprising 30 amino acid residues or less, the peptide having an amino acid sequence of the formula SEQ ID NO: 12,
Gly Lcu Lys Pro Gly Val Asp X1 Thr Ile Thr X2 X3 Ala X4
wherein
X1 represents Tyr or Ala;
X2 represents Val or Ala;
X3 represents Tyr or Ala; and
X4 represents Val or Ala.
Also, the invention relates to the physiologically active peptide described above (SEQ ID NO: 2) wherein X1 is Tyr, X2 is Val, X3 is Tyr, and X4 is Val.
Also, the invention relates to the physiologically active peptide described above (SEQ ID NO:3) wherein X1 is Ala, X2 is Val, X3 is Tyr, and X4 is Val.
Also, the invention relates to the physiologically active peptide described above (SEQ ID NO: 7) wherein X1 is Tyr, X2 is Ala, X3 is Tyr, and X4 is Val.
Also, the invention relates to the physiologically active peptide described above (SEQ ID NO: 8) wherein X1 is Tyr, X2 is Val, X3 is Ala, and X4 is Val.
Also, the invention relates to the physiologically active peptide described above (SEQ ID NO: 9) wherein X1 is Tyr, X2 is Val, X3 is Tyr, and X4 is Ala.
Further, the invention relates to an anti-metastatic of cancer containing any of the physiologically active peptides described above.
Also, the invention relates to the anti-metastatic of cancer described above wherein the physiologically active peptide is further conjugated with biotin.
The present invention will be more fully understood from the detailed description given hereinbelow and the accompanying drawings, which are given by way of illustration only and are not to be considered as limiting the present invention.
Further scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will be apparent to those skilled in the art from this detailed description.